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• W2000716940 abstract "The closely related mycobacteria responsible for tuberculosis produce an unusually high number of secreted proteins, many of which are clearly implicated in pathogenesis and protective immunity. Falling within this category are the closely related proteins MPB70 and MPB83. The structure of MPB70 reveals a complex and novel bacterial fold, which has clear structural homology to the two C-terminal FAS1 domains of the cell adhesion protein fasciclin I, whose structures were reported very recently. Assessment of the surface features of MPB70, the sequence divergence between MPB70 and MPB83, the conservation of residues across a group of FAS1 domains, and the locations of disease-inducing mutations in βig-h3 strongly suggests that MPB70 and MPB83 contain two functional surfaces on opposite faces, which are probably involved in binding to host cell proteins. This analysis also suggests that these functional surfaces are retained in the FAS1 proteins associated with mediating interactions between cells and the extracellular matrix (fasciclin I, periostin, and βig-h3) and furthermore that some of the human corneal disease-inducing substitutions identified in βig-h3 will perturb interactions at these sites. The closely related mycobacteria responsible for tuberculosis produce an unusually high number of secreted proteins, many of which are clearly implicated in pathogenesis and protective immunity. Falling within this category are the closely related proteins MPB70 and MPB83. The structure of MPB70 reveals a complex and novel bacterial fold, which has clear structural homology to the two C-terminal FAS1 domains of the cell adhesion protein fasciclin I, whose structures were reported very recently. Assessment of the surface features of MPB70, the sequence divergence between MPB70 and MPB83, the conservation of residues across a group of FAS1 domains, and the locations of disease-inducing mutations in βig-h3 strongly suggests that MPB70 and MPB83 contain two functional surfaces on opposite faces, which are probably involved in binding to host cell proteins. This analysis also suggests that these functional surfaces are retained in the FAS1 proteins associated with mediating interactions between cells and the extracellular matrix (fasciclin I, periostin, and βig-h3) and furthermore that some of the human corneal disease-inducing substitutions identified in βig-h3 will perturb interactions at these sites. Tuberculosis remains one of the most significant infectious diseases of humans, with about one-third of the world's population currently infected resulting in about 3 million deaths annually (1Murray C.J.L. Salomon J.A. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 13881-13886Crossref PubMed Scopus (215) Google Scholar, 2Young D.B. Robertson B.D. Science. 1999; 284: 1479-1480Crossref PubMed Scopus (12) Google Scholar). The bacteria responsible for tuberculosis belong to the Mycobacterium tuberculosis complex, which is a group of highly related mycobacteria. The complex includes M. tuberculosis, which causes the majority of human tuberculosis, and Mycobacterium bovis, which leads to tuberculosis in a range of domesticated and wild animals. Analysis of the complete M. tuberculosis H37Rv genome has identified the genes for 4006 proteins (3Cole S.T. Brosch R. Parkhill J. Garnier T. Churcher C. Harris D. Gordon S.V. Eiglmeier K. Gas S. Barry C.E. Tekaia F. Badcock K. Basham D. Brown D. Chillingworth T. Connor R. Davies R. Devlin K. Feltwell T. Gentles S. Hamlin N. Holroyd S. Hornsby T. Jagels K. Krogh A. McLean J. Moule S. Murphy L. Oliver K. Osborne J. Quail M.A. Rajandream M.-A. Rogers J. Rutter S. Seeger K. Skelton J. Squares R. Squares S. Sulston J.E. Taylor K. Whitehead S. Barrell B.G. Nature. 1998; 393: 537-544Crossref PubMed Scopus (6522) Google Scholar, 4Cole S.T. Eiglmeier K. Parkhill J. James K.D. Thomson N.R. Wheeler P.R. Honore N. Garnier T. Churcher C. Harris D. Mungall K. Basham D. Brown D. Chillingworth T. Connor R. Davies R.M. Devlin K. Duthoy S. Feltwell T. Fraser A. Hamlin N. Holroyd S. Hornsby T. Jagels K. Lacroix C. Maclean J. Moule S. Murphy L. Oliver K. Quail M.A. Rajandream M.-A. Rutherford K.M. Rutter S. Seeger K. Simon S. Simmonds M. Skelton J. Squares R. Squares S. Stevens K. Taylor K. Whitehead S. Woodward J.R. Barrell B.G. Nature. 2001; 409: 1007-1011Crossref PubMed Scopus (1370) Google Scholar), however, it has so far proved possible to assign specific functions to only about half of the predicted proteins. In addition, we still have relatively little information about which M. tuberculosis complex proteins are essential for pathogenesis, or associated with the stimulation of protective immunity, and even less knowledge of their structures, functions, and mechanisms of action. The finding that only live mycobacterial vaccines provide significant protection against tuberculosis infection (5Orme I.M. Infect. Immun. 1988; 56: 3310-3312Crossref PubMed Google Scholar) clearly indicates a key role for secreted M. tuberculosis complex proteins in stimulating protective immunity and highlights the importance of investigating major secreted antigenic proteins such as MPB70, MPB83, ESAT-6, and CFP-10 (6Hewinson R.G. Russell W.P. J. Gen. Microbiol. 1993; 139: 1253-1259Crossref PubMed Scopus (24) Google Scholar, 7Harboe M. Wiker H.G. Ulvund G. Lund-Pedersen B. Andersen A.B. Hewinson R.G. Nagai S. Infect. Immun. 1998; 66: 289-296Crossref PubMed Google Scholar, 8Berthet F.-X. Rasmussen P.B. Rosenkrands I. Andersen P. Gicquel B. Microbiology. 1998; 144: 3195-3203Crossref PubMed Scopus (422) Google Scholar, 9Bloemink M.J. Kemmink J. Dentten E. Muskett F.W. Whelan A. Sheikh A. Hewinson G. Williamson R.A. Carr M.D. J. Biomol. NMR. 2001; 20: 185-186Crossref PubMed Scopus (2) Google Scholar, 10Renshaw P.S. Panagiotidou P. Whelan A. Gordon S.V. Hewinson R.G. Williamson R.A. Carr M.D. J. Biol. Chem. 2002; 277: 21598-21603Abstract Full Text Full Text PDF PubMed Scopus (264) Google Scholar). The mature M. bovis protein MPB70 and its identical M. tuberculosis homologue MPT70 (Rv2875) are stable, 163-residue polypeptides, that are efficiently secreted from mycobacterial cells following cleavage of a 30-residue signal peptide (3Cole S.T. Brosch R. Parkhill J. Garnier T. Churcher C. Harris D. Gordon S.V. Eiglmeier K. Gas S. Barry C.E. Tekaia F. Badcock K. Basham D. Brown D. Chillingworth T. Connor R. Davies R. Devlin K. Feltwell T. Gentles S. Hamlin N. Holroyd S. Hornsby T. Jagels K. Krogh A. McLean J. Moule S. Murphy L. Oliver K. Osborne J. Quail M.A. Rajandream M.-A. Rogers J. Rutter S. Seeger K. Skelton J. Squares R. Squares S. Sulston J.E. Taylor K. Whitehead S. Barrell B.G. Nature. 1998; 393: 537-544Crossref PubMed Scopus (6522) Google Scholar, 6Hewinson R.G. Russell W.P. J. Gen. Microbiol. 1993; 139: 1253-1259Crossref PubMed Scopus (24) Google Scholar). The proteins contain a single disulfide bond linking Cys-8 and Cys-142 but show no other form of post-translational modification. MPB70 is a major serodominant antigen of M. bovis, which also stimulates cellular immune responses during infection and is able to elicit a strong, delayed type hypersensitivity response in M. bovis infected cattle (11Billman-Jacobe H. Radford A.J. Rothel J.S. Wood P.R. Immunol. Cell Biol. 1990; 68: 359-365Crossref PubMed Google Scholar, 12Fifis T. Costopoulos C. Radford A.J. Bacic A. Wood P.R. Infect. Immun. 1991; 59: 800-807Crossref PubMed Google Scholar, 13Fifis T. Corner L.A. Rothel J.S. Wood P.R. Scand. J. Immunol. 1994; 39: 267-274Crossref PubMed Scopus (59) Google Scholar, 14Pollock J.M. Douglas A.J. MacKie D.P. Neill S.D. Immunology. 1994; 82: 9-15PubMed Google Scholar). Virulent M. bovis strains secrete high levels of MPB70 when grown in culture, but the protein is produced at much lower levels by M. tuberculosis and by a number of M. bovis BCG strains including Pasteur, Copenhagen, and Glaxo (7Harboe M. Wiker H.G. Ulvund G. Lund-Pedersen B. Andersen A.B. Hewinson R.G. Nagai S. Infect. Immun. 1998; 66: 289-296Crossref PubMed Google Scholar, 15Miura K. Nagai S. Kinomoto M. Haga S. Tokunaga T. Infect. Immun. 1983; 39: 540-545Crossref PubMed Google Scholar, 16Harboe M. Nagai S. Am. Rev. Respir. Dis. 1984; 129: 444-452PubMed Google Scholar). Despite significant differences in expression levels in culture both M. bovis and M. tuberculosis stimulate a strong immune response to MPB70 upon infection, suggesting that expression of the protein is up-regulated by M. tuberculosis in vivo (17Roche P.W. Triccas J.A. Avery D.T. Fifis T. Billman-Jacobe H. Britton W.J. J. Infect. Dis. 1994; 170: 1326-1330Crossref PubMed Scopus (67) Google Scholar, 18Hewinson R.G. Michell S. Russell W.P. McAdam R.A. Jacobs W.R. Scand. J. Immunol. 1996; 43: 490-499Crossref PubMed Scopus (109) Google Scholar). In addition, the treatment of M. tuberculosis-infected mice with a DNA vaccine encoding MPB70 has a pronounced therapeutic affect, indicating an essential role for MPB70 during infection of the host (19Lowrie D.B. Tascon R.E. Bonato V.L.D. Lima V.M.F. Faccioli L.H. Stavropoulos E. Colston M.J. Hewinson R.G. Moelling K. Silva C.L. Nature. 1999; 400: 269-271Crossref PubMed Scopus (417) Google Scholar). Although MPB70 is clearly linked to the stimulation of protective immunity and implicated in tuberculosis pathogenesis, the function of the protein remains unknown. Interestingly, residues 26–162 from MPB70 show significant sequence homology (up to 30% identity) to the FAS1 domains of several extracellular matrix proteins (fasciclin I, βig-h3, and periostin), which are known to be involved in mediating interactions between cells and the extracellular matrix (20Kim J.-E. Kim S.-J. Lee B.-H. Park R.-W. Kim K.-S. Kim I.-S. J. Biol. Chem. 2000; 275: 30907-30915Abstract Full Text Full Text PDF PubMed Scopus (240) Google Scholar, 21Billings P.C. Whitbeck J.C. Adams C.S. Abrams W.R. Cohen A.J. Engelsberg B.N. Howard P.S. Rosenbloom J. J. Biol. Chem. 2002; 277: 28003-28009Abstract Full Text Full Text PDF PubMed Scopus (113) Google Scholar). Mycobacteria of the M. tuberculosis complex produce another major secreted protein termed MPB83 in M. bovis and MPT83 (Rv2873) in M. tuberculosis (identical sequences), which is highly homologous to MPB70 or MPT70 (Rv2875) and as with MPB70 appears to stimulate a strong protective immune response upon infection (18Hewinson R.G. Michell S. Russell W.P. McAdam R.A. Jacobs W.R. Scand. J. Immunol. 1996; 43: 490-499Crossref PubMed Scopus (109) Google Scholar, 22Morris S. Kelley C. Howard A. Li Z. Collins F. Vaccine. 2000; 18: 2155-2163Crossref PubMed Scopus (124) Google Scholar, 23Chambers M.A. Williams A. Hatch G. Gavier-Widen D. Hall G. Huygen K. Lowrie D. Marsh P.D. Hewinson R.G. Infect. Immun. 2002; 70: 2159-2165Crossref PubMed Scopus (46) Google Scholar). The genes encoding MPB/MPT70 and MPB/MPT83 appear to form part of a larger operon (Rv2871 to Rv2875 in M. tuberculosis) and are expressed under identical conditions (7Harboe M. Wiker H.G. Ulvund G. Lund-Pedersen B. Andersen A.B. Hewinson R.G. Nagai S. Infect. Immun. 1998; 66: 289-296Crossref PubMed Google Scholar, 18Hewinson R.G. Michell S. Russell W.P. McAdam R.A. Jacobs W.R. Scand. J. Immunol. 1996; 43: 490-499Crossref PubMed Scopus (109) Google Scholar, 24Dolores M. Ascencion J. Espita C. FEMS Microbiol. Lett. 2001; 203: 95-102Crossref PubMed Google Scholar). The mature secreted form of MPB83 contains 196 residues, with residues 33–195 over 80% homologous to full-length mature MPB70 (74% identity over 163 residues). On the basis of sequence conservation MPB83 is expected to contain a single disulfide bond analogous to that found in MPB70 linking Cys-40 and Cys-174, but the protein is also glycosylated on T24 and T25 and lipoylated at the N terminus (25Michell S.L. Whelan A.O. Wheeler P.R. Panico M. Easton R.L. Etienne A.T. Haslam S.M. Dell A. Morris H.R. Reason A.J. Herrmann J.L. Young D.B. Hewinson R.G. J. Biol. Chem. 2003; 278: 16423-16432Abstract Full Text Full Text PDF PubMed Scopus (72) Google Scholar). As with MPB70, the function of MPB83 is not known, however, in contrast to MPB70 it is localized to the surface of M. tuberculosis complex cells and anchored to the cell wall via the N-terminal lipid group (7Harboe M. Wiker H.G. Ulvund G. Lund-Pedersen B. Andersen A.B. Hewinson R.G. Nagai S. Infect. Immun. 1998; 66: 289-296Crossref PubMed Google Scholar), which suggests that the two closely related proteins have evolved distinct functional roles. In this communication we report the determination of the high resolution solution structure of MPB70, which contains a complex fold not previously observed in bacterial proteins and shows close structural homology to the third and fourth FAS1 domains of Drosophila fasciclin I whose structures were reported during the preparation of the manuscript for this report (26Clout N.J. Tisi D. Hohenester E. Structure. 2003; 11: 197-203Abstract Full Text Full Text PDF PubMed Scopus (92) Google Scholar). The structure obtained for MPB70 has allowed us to assess the functional implications of surface features on the protein and of the sequence divergence between MPB70 and MPB83. In addition, the MPB70 and fasciclin I structures have enabled us to examine the roles of residues conserved across a group of FAS1 domain-containing proteins and to consider the functional and structural consequences of mutations in the fourth FAS1 domain of human βig-h3, which lead to severe impairment of vision through progressive degeneration of the cornea (inherited corneal dystrophies (27Munier F.L. Frueh B.E. Othenin-Girard P. Uffer S. Cousin P. Wang M.X. Heon E. Black G.C.M. Blasi M.A. Balestrazzi E. Lorenz B. Escoto R. Barraquer R. Hoeltzenbein M. Gloor B. Fossarello M. Singh A.D. Arsenijevic Y. Zografos L. Schorderet D.F. Invest. Ophthalmol. Vis. Sci. 2002; 43: 949-954PubMed Google Scholar)). Sample Preparation—The 15N- and 15N/13C-labeled MPB70 were prepared from a pBluescript KS+-based Escherichia coli expression vector (pVW500) as described previously (6Hewinson R.G. Russell W.P. J. Gen. Microbiol. 1993; 139: 1253-1259Crossref PubMed Scopus (24) Google Scholar, 9Bloemink M.J. Kemmink J. Dentten E. Muskett F.W. Whelan A. Sheikh A. Hewinson G. Williamson R.A. Carr M.D. J. Biomol. NMR. 2001; 20: 185-186Crossref PubMed Scopus (2) Google Scholar). The expression system produces the mature form of MPB70 (163 residues) as a secreted product, which is purified from the culture supernatant and periplasmic cell fraction. The majority of the MPB70 samples were either uniformly 15N- or 15N/13C-labeled, however, 13C/1H HMQC-NOESY 1The abbreviations used are: HMQC, heteronuclear multiple quantum coherence; NOESY, nuclear Overhauser effect spectroscopy; TOCSY, total correlation spectroscopy; CANDID, combined automated NOE assignment and structure determination protocol; r.m.s.d., root mean square deviation; CE, combinatorial extension; DQF, double quantum filtered. spectra were acquired from MPB70 in which only the non-aromatic residues were labeled. This was achieved by preparing the protein from cells grown on labeled minimal medium (2 g/liter [15N]ammonium sulfate and 5.5 g/liter [13C]glucose) supplemented with 50 mg/liter unlabeled l-histidine, l-tyrosine, and l-phenylalanine. The NMR experiments were carried out on 0.35-ml samples of 1.0 mm15N-, 0.7 mm uniformly 15N/13C-, and 1.2 mm non-aromatic residue 15N/13C-labeled MPB70 in 20 mm sodium phosphate buffer (pH 6.0) containing 100 mm sodium chloride and either 90% H2O/10% D2O or 100% D2O as appropriate. NMR Spectroscopy—All the NMR spectra were acquired at 30 °C on either 600- or 800-MHz Varian Inova spectrometers. The two- and three-dimensional spectra recorded to extend essentially complete sequence-specific backbone resonance assignments for MPB70 to the side chains and to obtain conformational constraints for structural calculations were: 1H DQF-COSY (28Rance M. Sorensen O.W. Bodenhausen G. Wagner G. Ernst R.R. Wüthrich K. Biochem. Biophys. Res. Commun. 1983; 117: 479-485Crossref PubMed Scopus (2597) Google Scholar) and TOCSY using a 10-kHz MLEV17-based mixing time of 45 ms (29Braunschweiler L. Ernst R.R. J. Magn. Reson. 1983; 53: 521-528Crossref Scopus (3108) Google Scholar), 15N/1H HSQC, TOCSY-HSQC with mixing periods of 28 and 58 ms, NOESY-HSQC using a NOE mixing time of 100 ms (30Marion D. Kay L.E. Sparks S.W. Torchia D.A. Bax A. J. Am. Chem. Soc. 1989; 111: 1515-1517Crossref Scopus (593) Google Scholar), HNHA (31Vuister G.W. Bax A. J. Am. Chem. Soc. 1993; 115: 7772-7777Crossref Scopus (1051) Google Scholar) and HNHB (32Archer S.J. Ikura M. Torchia D.A. Bax A. J. Magn. Reson. 1991; 95: 636-641Google Scholar), and 13C/1H HCCH-TOCSY with a 7.5-kHz DIPSI-3-based mixing period of 14 ms (33Bax A. Clore G.M. Gronenborn A.M. J. Magn. Reson. 1990; 88: 425-431Google Scholar) and HMQC-NOESY using a NOE mixing time of 100 ms (34Zuiderweg E.R.P. McIntosh L.P. Dahlquist F.W. Fesik S.W. J. Magn. Reson. 1990; 86: 210-216Google Scholar). The three-dimensional spectra were acquired over about 85 h with typical maximum evolution times in F1 or F2 of 12–14 ms for 15N, 6–10 ms for 13C, and 15–18 ms for 1H and acquisition times in F3 of 50–150 ms. The decoupling of 15N and 13C from 1H during the acquisition time was usually achieved using a GARP1 sequence, but at 800 MHz 1H adiabatic WURST sequences were used for 13C decoupling (35Kupce E. Freeman R. J. Magn. Reson. A. 1996; 118: 299-303Crossref Scopus (182) Google Scholar). The two-dimensional 1H spectra were recorded over about 40 h with acquisitions times of 35–40 ms in F1 and 250 ms in F2. In all experiments the WATER-GATE method was used to suppress the water signal when required (36Sklenar V. Piotto M. Leppik R. Saudek V. J. Magn. Reson. 1993; 102: 241-245Crossref Scopus (1112) Google Scholar). The three-dimensional NMR data were processed using NMRPipe (37Delaglio F. Grzesiek S. Vuister G.W. Zhu G. Pfeifer J. Bax A. J. Biomol. NMR. 1995; 6: 277-293Crossref PubMed Scopus (11570) Google Scholar), with linear prediction used to extend the effective acquisition times by up to 1.5-fold in F1 and F2 and mild resolution enhancement applied in all dimensions using a shifted sine-squared function. Apart from the omission of linear prediction, the two-dimensional spectra were similarly processed using the Varian VNMR package. All the spectra were analyzed on-screen using the program XEASY (38Bartels C. Xia T. Billeter M. Güntert P. Wüthrich K. J. Biomol. NMR. 1995; 5: 1-10Crossref PubMed Scopus (1604) Google Scholar). Structural Calculations—The family of converged MPB70 structures was calculated in a two-stage process using the program CYANA (39Güntert P. Mumenthaler C. Wütrich K. J. Mol. Biol. 1997; 273: 283-298Crossref PubMed Scopus (2555) Google Scholar). Initially, the recently published combined automated NOE assignment and structure determination protocol (CANDID) was used to automatically assign the NOE cross-peaks identified in three-dimensional 15N- and 13C-edited NOESY spectra of MPB70 and to produce preliminary structures of the protein (40Herrmann T. Güntert P. Wütrich K. J. Mol. Biol. 2002; 319: 209-227Crossref PubMed Scopus (1329) Google Scholar). After which several cycles of simulated annealing combined with redundant dihedral angle constraints to increase convergence were used to produce the final converged MPB70 structures (41Güntert P. Wütrich K. J. Biomol. NMR. 1991; 1: 447-456Crossref PubMed Scopus (336) Google Scholar). The input for the CANDID stage primarily consisted of essentially complete 15N, 13C, and 1H resonance assignments for the non-exchangeable groups in MPB70 and three manually picked three-dimensional NOE peak lists corresponding to all NOEs involving amide protons (1589 peaks), NOEs between aliphatic protons and aromatic side chain or slowly exchanging backbone amide protons (750 peaks), and all NOEs between aliphatic protons (3434 peaks). In addition, the CANDID stage included constraints corresponding to the single disulfide bond present in MPB70 (Cys-8 to Cys-142) and to 35 χ1 angles (±30°) derived from analysis of TOCSY-HSQC and HNHB spectra. The NOE peak lists were prepared using XEASY, and the volumes of the cross-peaks were calculated using the Lorentzian line fitting and integration routines available in SPSCAN (www.molebio.uni-jena.de/wg/spscan). The CANDID calculations were carried out using the default parameter settings in CYANA 1.05 apart from increasing the average target value for NOEs involving backbone protons from 3.8 to 4.0 Å in the structure-independent NOE calibration routine used in cycle 1, raising the upper limit for observable NOEs to 6.0 Å and setting the chemical shift uncertainties to 0.02 ppm for 1H, 0.3 ppm for 15N, and 0.2 ppm for 13C. The final converged MPB70 structures were produced from 100 random starting coordinates using a torsion angle-based simulated annealing protocol combined with four cycles of redundant dihedral angle constraints, essentially as described previously (42Muskett F.W. Frenkiel T.A. Feeney J. Freedman R.B. Carr M.D. Williamson R.A. J. Biol. Chem. 1998; 274: 37226-37232Google Scholar, 43Lemercinier X. Muskett F.W. Cheeseman B. McIntosh P.B. Thim L. Carr M.D. Biochemistry. 2001; 40: 9552-9559Crossref PubMed Scopus (20) Google Scholar). The calculations were mainly based upon 2860 non-redundant, NOE-derived upper distance limits, assigned to unique pairs of protons using CANDID but also included 35 χ1 angle constraints and appropriate upper and lower distance limits corresponding to the Cys-8 to Cys-142 disulfide bond, and in the final round of the calculations to 45 hydrogen bonds involving backbone amide groups. The hydrogen bond constraints were only included for residues with backbone amide signals still detectable after 8 weeks in D2O and where the distance between the hydrogen bond acceptor and donor atoms was less than 2.5 Å and the NH to O bond angle was greater than 135° in converged structures obtained from the penultimate calculation cycle (residues 40, 41, 43–49, 51, 55, 61, 66–70, 72, 87, 98–103, 105, 114, 116, 118, 119, 121, 124, 126, 127, 134, 135, 140, 141, 145, 147, 151, 153–155, and 159). The standard simulated annealing protocol used consisted of a high temperature conformational search phase of 2000 steps followed by slow cooling over 8000 steps and conjugate gradient minimization at the end. The family of MPB70 structures obtained were analyzed using the programs CYANA (39Güntert P. Mumenthaler C. Wütrich K. J. Mol. Biol. 1997; 273: 283-298Crossref PubMed Scopus (2555) Google Scholar) and MOLMOL (44Koradi R. Billeter M. Wüthrich K. J. Mol. Graphics. 1996; 14: 51-55Crossref PubMed Scopus (6490) Google Scholar). An exhaustive similarity search of the MPB70 coordinates against all structures deposited in the Protein Data Bank was carried out using Dali (Ref. 45Holm L. Sander C. Trends Biochem. Sci. 1995; 20: 478-480Abstract Full Text PDF PubMed Scopus (1283) Google Scholar; available at www.ebi.ac.uk/dali), and optimal alignments between the structures for MPB70 and the fasciclin domains of fasciclin I were calculated using CE (Ref. 46Shindyalov I.N. Bourne P.E. Protein Eng. 1998; 11: 739-747Crossref PubMed Scopus (1701) Google Scholar; available at cl.sdsc.edu/ce). Sequence-specific Assignments—The essentially complete sequence-specific 15N, 13C, and 1H backbone resonance assignments reported for MPB70 previously (9Bloemink M.J. Kemmink J. Dentten E. Muskett F.W. Whelan A. Sheikh A. Hewinson G. Williamson R.A. Carr M.D. J. Biomol. NMR. 2001; 20: 185-186Crossref PubMed Scopus (2) Google Scholar) were readily extended to the 13C and 1H side-chain signals using correlations observed in DQF-COSY, TOCSY, TOCSY-HSQC, and HCCH-TOCSY spectra. Apart from exchangeable side-chain groups, the only 1H signals that remained unidentified were from the backbone amides of G1 and D2 and from the Hγ of the side chain of Leu-119. The extent of the assignments obtained for 13C resonances was equally comprehensive with just 11 residues lacking complete assignments for the aliphatic signals (Gly-1, Leu-3, Thr-41, Thr-42, Val-55, Leu-94, Thr-107, Leu-119, Leu-133, Ile-155, and Pro-161), with resonances from typically only a single group remaining unidentified. In the case of MPB70, no attempts were made to assign the 13C signals of the aromatic rings, because this information was not required for the analysis of the 13C-edited NOESY spectrum, which was acquired from a sample of MPB70 in which the side chains of the aromatic residues were not 13C-labeled. Structural Calculations—The recently developed CANDID protocol proved very effective at determining unique assignments for the NOEs identified in three-dimensional 15N- and 13C-edited NOESY spectra (40Herrmann T. Güntert P. Wütrich K. J. Mol. Biol. 2002; 319: 209-227Crossref PubMed Scopus (1329) Google Scholar). At the end of the procedure (cycle 7) unique assignments were obtained for 94.3% (1498/1589) of the NOE peaks picked in the 15N/1H NOESY-HSQC spectrum and 96.3% (4030/4184) of those identified in the 13C/1H HMQC-NOESY spectrum. This level of success compares very favorably with our experience of manual iterative assignment of NOE peaks in protein spectra (42Muskett F.W. Frenkiel T.A. Feeney J. Freedman R.B. Carr M.D. Williamson R.A. J. Biol. Chem. 1998; 274: 37226-37232Google Scholar, 43Lemercinier X. Muskett F.W. Cheeseman B. McIntosh P.B. Thim L. Carr M.D. Biochemistry. 2001; 40: 9552-9559Crossref PubMed Scopus (20) Google Scholar, 47McIntosh P.B. Frenkiel T.A. Wollborn U. McCormick J.E. Klempnauer K.-H. Feeney J. Carr M.D. Biochemistry. 1998; 37: 9619-9629Crossref PubMed Scopus (24) Google Scholar), and the automatic approach took only a few days as compared with several months for manual assignment. The uniquely assigned NOE peaks produced 2845 non-redundant 1H to 1H upper distance limits, which were used as the principle constraints in the final rounds of structural calculations. In contrast to the success in assigning NOEs, CANDID was relatively poor at producing MPB70 structures with no significant violations of NMR constraints or van der Waals interactions, with typically only one or two fully converged structures obtained from 100 random starting coordinates. This convergence problem may well reflect the complex topology of the MPB70 backbone and was solved by the inclusion of redundant dihedral angle constraints in the final rounds of simulated annealing calculations (41Güntert P. Wütrich K. J. Biomol. NMR. 1991; 1: 447-456Crossref PubMed Scopus (336) Google Scholar). The final family of MPB70 structures was determined using a total of 3066 NMR-derived structural constraints (an average of 18.8 per residue), including 2845 NOE-based upper distance limits (542 intra residue, 733 sequential (i, i+1), 562 medium range (i, i ≤ 4), and 1008 long range (i, i ≥ 5)) and 35 χ1 torsion angle constraints. The calculations also included 180 distance constraints required to impose 45 backbone hydrogen bonds and 6 distance constraints corresponding to the Cys-8 to Cys-142 disulfide bond. After the final round of CYANA calculations, 39 satisfactorily converged MPB70 structures were obtained from 100 random starting conformations. The converged structures contain no distance constraint or van der Waals violations greater than 0.5 Å and no dihedral angle violations greater than 5°, with an average value for the CYANA target function of 9.5 ± 1.3. The sums of the violations for upper distance limits, lower distance limits, van der Waals contacts, and torsion angle constraints were 39.8 ± 3.3 Å, 2.1 ± 0.3 Å, 18.3 ± 2.1 Å, and 11.7 ± 3.8°, respectively. Similarly, maximum violations for the converged structures were 0.39 ± 0.05 Å, 0.22 ± 0.03 Å, 0.30 ± 0.03 Å, and 3.35 ± 0.90°, respectively. The family of converged MPB70 structures, together with the NMR constraints, has been deposited in the Protein Data Bank (accession code 1NYO). The solution structure of MPB70 is determined to high precision, which is clearly evident from the superposition of the protein backbone shown for the family of converged structures in Fig. 1 (best fit for residues 3–128 and 132–162) and is reflected in low root mean squared deviation (r.m.s.d.) values to the mean structure for both the backbone and all heavy atoms of 0.43 ± 0.05 Å and 0.71 ± 0.05 Å, respectively. A few N- and C-terminal residues (Gly-1, Asp-2, and Ala-163), together with a short surface loop formed from Gln-129 to Asn-131, showed no long range NOEs and medium range NOEs only in the case of Gln-129, which results in the conformations of these regions being somewhat less well defined, probably as a consequence of rapid local mobility. The MPB70 structures show good non-bonded contacts, with 62% of the non-glycine and non-proline residues found to have backbone torsion angles in the most favored regions, 33% in additional allowed regions, 3% in generously allowed regions, and only 1% in disallowed regions. In addition, no residues consistently adopt unfavorable backbone conformations. Structural Features of MPB70 —The backbone topology of MPB70 is illustrated by the ribbon diagram shown in Fig. 2, with the protein consisting of a seven-stranded β-barrel (β1 66–71, β2 102–104, β3 114–117, β4 123–128, β5 133–135, β6 138–147, and β7 150–155) and eight α-helices (α1 7–14, α2 22–27, α331–36, α441–48, α558–61, α673–78, α781–87, and α8 93–100) that pack together on one side of the barrel. The β-barrel is closed by one hydrogen bond between β2 and β3, and so, it alternatively can be thought of as a β-sandwich with strand 6 coiling to form part of two sheets (β6C-β7-β1-β2 and β3-β4-β5-β6N). All the strands are anti-parallel to each other except β1 and β7, and the residue offset for one transverse around the barrel is 10 (the shear number). As shown in Fig. 2, the circumference of the barrel is wider at the" @default.
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• W2000716940 date "2003-10-01" @default.
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• W2000716940 title "Solution Structure of the Mycobacterium tuberculosis Complex Protein MPB70" @default.
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• W2000716940 doi "https://doi.org/10.1074/jbc.m307235200" @default.
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